Methylmercury (MeHg) is a prominent environmental neurotoxicant that induces cerebellar toxicity including degeneration of granule neurons. We hypothesize that chronic exposure to MeHg induces a primary elevation of intracellular Ca2+ ([Ca2+]i) which triggers a cascade of effects including disruption of membrane excitability, altered neurotransmitter secretion, impaired macromolecule synthesis, and ultimately cell death. Proposed studies in this renewal application will focus on the relationship of MeHg-induced changes in [Ca2+]i to effects on ionic channels, synaptic function and alteration of Ca2+-dependent neuronal signalling in response to chronic toxicity, especially of cerebellar granule neurons. Studies will utilize in vitro brain slice preparations from rats, cultured granule neurons or astrocytes, or rat Pc12 pheochromocytoma cells. Effects of acute and chronic exposure to MeHg will be examined using a combination of electrophysiological, biochemical, and digital imaging determinations of fluorescent indicator dyes of Ca2+ concentrations. Specific questions include: 1) Is influx of Ca2+ and its subsequent buffering perturbed following chronic treatment with MeHg (Can these changes be linked to changes in excitability and/or synaptic function and do they result in altered levels of signal transduction molecules)? 2( Do cerebellar granule neurons differ in their susceptibility to MeHg with respect to [Ca2+]i buffering and Ca2+ ion channel conductances compared to astroglia or other neurons? 3) Does MeHg alter Ca2+ channel function of neurons or glia directly or secondarily due to indirect actions such as elevation of [Ca2+]i, depolarization of the membrane channel, phosphorylation or activation of G-proteins? 4) By elevating [Ca2+]i, does MeHg induce alteration of currents carried across the membrane by nonspecific cation channels or certain types of K+ channels? 5) Does MeHg disrupt cerebellar transmission at granule cell-Purkinje cell synapses? In particular, are dendritic Ca2+-dependent action potentials disrupted? 6) Is synaptic transmission in cerebellar slices affected preferentially or differentially by MeHg compared to other brain regions such as hippocampus, which are less affected during intoxication? Intracellular microelectrode recordings and patch voltage clamp recordings will be made from cerebellar granule/Purkinje synapses in isolated slices of rat brain. Effects of MeHg on release of transmitter and iontophoretic responses to glutamate as well as on Ca2+-driven action potentials will be measured. Effects of MeHg on currents carried through voltage- dependent Ca channels as well as K+ and nonspecific cation channels activated by Ca will be examined in single cells in culture or single cells in slices using patch voltage clamp. Spatial and temporal changes of [Ca]i in response to MeHg will be examined using digital imaging of fura 2-mediated fluorescence alone or in combination with patch-clamp recordings of single cells to test the relationship between changes in ion channel activity and elevation of [Ca2+]i. Alterations by MeHg of [Na+]i will be studied using the fluorescent indicator SBFI to determine if MeHg causes a generalized increase in intracellular cations. Results of these studies should lead to better understanding of a) potential sites perturbed after chronic exposure to MeHg, particularly the relationship of early acute actions on membrane and [Ca2+]i handling to chronic effects on macromolecule synthesis and neuronal death, and b) explanations for heightened susceptibility of cerebellar granule neurons to MeHg.